Antibody against aflatoxins, support using the antibody, method of immunologically detecting aflatoxins and method of concentrating and purifying aflatoxins

ABSTRACT

It is intended to detect, concentrate and purify aflatoxins of all types which are possibly contained in a sample such as a food. It is also intended to detect the total amount or the individual amounts thereof at a high sensitivity. By using aflatoxin B2 or its derivative as a hapten compound, an antibody, which shows the same reactivity to individual aflatoxin analogs and is highly tolerant to organic solvents, is obtained. Then, a detection/concentration/purification means and an immunological detection means with the use of the above antibody are constructed. The detection means thus constructed achieves a high sensitivity and excellent quantification properties.

TECHNICAL FIELD

The present invention relates to an antibody having novel propertiestoward aflatoxins or a fragment thereof. The present invention alsorelates to a support for capturing aflatoxins, an affinity column, amethod of immunologically detecting aflatoxins, and a method ofconcentrating and purifying aflatoxins, which use such an antibody orfragment thereof.

BACKGROUND ART

Aflatoxins that are natural toxins produced by fungi are known ascarcinogenetic toxins and can be detected in many foods such asprocessed goods including peanuts, peanut butter and the like, cerealsand processed goods thereof including corn, oats, buckwheat flour andthe like, spices including nutmeg, white pepper and the like, andpistachio nuts. Aflatoxins are secondary metabolites produced byfilamentous bacteria such as Aspergillus flavus and Aspergillusparasiticus. Aflatoxin analogs such as B1, B2, G1, G2 are known.Further, a water-soluble aflatoxin metabolite M1, found in milk fromcattle having ingested aflatoxins, has been isolated and characterized.It is widely known that among them, aflatoxin B1 when orally ingestedexhibits extremely high carcinogenicity.

Structural analogs such as aflatoxins B2, G1, G2 and M1 other thanaflatoxin B1, although varying in carcinogenicity more or less dependingon animal species and having not so strong toxicity as that of B1, arealso recognized to be carcinogenic and acutely toxic in various animals.

In Japan, the concentration of aflatoxin B1 in food is regulated to be10 ppb or less by the Food Hygiene Law, and peanuts, corn and grainswherein A. flavus or aflatoxin B1 are detected with high frequency andwhich are imported from countries considered as contaminated territoryare particularly strictly examined. Because it is necessary that a largenumber of samples be rapidly dealt with, and a very small amount ofaflatoxin be detected, this examination makes use of thin layerchromatography (TLC), an HPLC fluorescence method, and in confirmationexamination, an LC/MS method.

When these chemical analysis methods are used, it is necessary that asample be pulverized and then extracted with an organic solvent,followed by concentrating and purifying its extract. As a concentrationand purification means, liquid-liquid partition, silica gel columnchromatography and Florisil column chromatography are usually used.Further, because the concentration and purification efficacy of suchchromatography is relatively low, antibody affinity columnchromatography utilizing the ability of an anti-aflatoxin and theantibody to bind to aflatoxins have come to be used in order to detect avery small amount of contaminating aflatoxins. However, the existingantibody column chromatography is susceptible to some organic solvents,and is thus disadvantageously limited in that only some organic solventsare usable and that said organic solvents may be usable only at lowconcentrations. Although many antibodies specific to highly toxicaflatoxin B1 have been developed and published, no antibody that alsoreacts nearly equally to aflatoxins B2, G1, G2, and M1 has been obtainedso far. Because aflatoxins B2, G1, G2, and M1 also have toxicities,including carcinogenicity, not only the content of aflatoxin B1 but alsothe individual contents and total amount of all aflatoxins are oftensubject to foreign regulations in recent years, and this tendency isestimated to increase from now. However, although there is a great needfor monoclonal antibodies against aflatoxins other than B1 type, theirstudy has been less advanced.

By way of example, previously known antibodies against aflatoxin B1poorly recognize other types of aflatoxins. Further, some monoclonalantibodies recognize aflatoxins B1, B2 and M1 only (for example,Non-Patent Documents 1 and 2). Further still, some monoclonal antibodiesrecognize highly toxic B1 and G1 only, but do not recognize other types(for example, Patent Document 1). Further still, antibodies formed usingB1 as a hapten may be less active toward G1; their ability to bind to G1may be reduced by several orders of magnitude relative to B1 or B2(Patent Document 2). On the other hand, there are monoclonal antibodiesuseful for detection of all aflatoxins (Patent Document 3), but thecrossreactivity among aflatoxins B1, B2, G1 and G2, when compared interms of IC50, varies greatly from ⅕ to 1/10 or less, and thus thesemonoclonal antibodies when used actually in detection of samples showvariation among the aflatoxin analogs.

Meanwhile, a method of quantifying aflatoxin B1 by using a monoclonalantibody against aflatoxin B1 has been studied (Non-Patent Document 3),and detection of aflatoxin B1 has been conducted by ELISA (enzyme-linkedimmunosorbent assay) etc. However, such a method is also disadvantageousin that since the antibody is susceptible to the organic solvent andreacts poorly with other analogs, the sample must be sufficientlydiluted similar to the level of dilution in antibody affinity columnchromatography and therefore, depending on the content ratio ofaflatoxin analogs in samples, aflatoxin B1 in some samples cannot beaccurately quantified.

-   Patent Document 1: U.S. Pat. No. 4,835,100-   Patent Document 2: JP-A 63-219394-   Patent Document 3: JP-A 4-360695-   Non-Patent Document 1: Candlish J. E. et al., Letters in Appl.    Microbiol. 1, 57 (1985)-   Non-Patent Document 2: Groopman et al., Proc. Natl. Acad. Sci. USA    81, 7728 (1984)-   Non-Patent Document 3: Hertzog P. J. et al., Carcinogenesis 3: pp.    825-828 (1982)

DISCLOSURE OF THE INVENTION Problems to be solved by the invention

As described above, the antibody against aflatoxins, althoughconventionally required to measure 4 types of oil-soluble aflatoxins and1 type of metabolite, has a disadvantage that the antibody issusceptible to some organic solvents and does not react equally react toeach analog. Further, antibody affinity column chromatography methodusing such antibodies is also susceptible to some organic solvents andcannot concentrate and purify all the analogs and metabolite equally.Moreover, the immunological measurement methods using such antibodiesare disadvantageous in that extracted samples must be sufficientlydiluted, and depending on the content ratio of aflatoxin analogs insample, the aflatoxin analogs in some samples cannot be accuratelyquantified. Accordingly, there has been demand for a series of methodssolving these problems.

Means for Solving the Problems

The present inventors have done extensive studies to provide a detectionmethod in which all types of aflatoxins, that is, aflatoxins B1, B2, G1,G2, and M1 can be equally detected, and even aflatoxins present ininfinitesimally small amounts of less than 10 ppb can also be accuratelydetected, and as a result, have found an antibody or fragment thereof,which has an ability to equally bind to all of these types of aflatoxinsand has organic solvent tolerance, as well as a detection method usingthe same, etc., and have completed the present invention.

That is, the antibody or fragment thereof of the present invention is anantibody or fragment thereof which is obtained by using an antigenderived from aflatoxin B2 or derivative thereof, has an ability toequally bind to all aflatoxins B1, B2, G1, G2, and M1, and has organicsolvent tolerance.

In one aspect of the present invention, the derivative of aflatoxin B2is a compound having a structure represented by the following formula(1):

In one aspect of the invention, the antigen derived from the derivativeof aflatoxin B2 is a conjugate between the compound of the formula (1)and a high-molecular weight compound.

The antibody described above may be a polyclonal antibody.

The antibody described above may be a monoclonal antibody.

The monoclonal antibody described above is AFB2-3-7F3-3.

The hybridoma of the present invention produces any of the monoclonalantibodies described above.

The hybridoma of the present invention may be a hybridoma which has beendeposited internationally under Accession No. FERM ABP-10931 since Nov.5, 2007, with International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology, Japan.

The method of immunologically detecting aflatoxins according to thepresent invention uses all of the antibody or any fragment thereofdescribed above.

The method of immunologically detecting aflatoxins according to thepresent invention can detect the amount of a single member or acombination of two or more members of aflatoxins B1, B2, G1, G2, and M1,or the total amount of all the members, by using any of the antibody orfragment thereof described above.

In one aspect of the method of immunologically detecting aflatoxinsaccording to the present invention, a solid phase adsorbent is furtherused.

The support of the present invention includes a solid phase adsorbentand any of the above antibody or fragment thereof immobilized on thesolid phase adsorbent.

The solid phase adsorbent described above may be Sepharose.

The affinity column of the present invention includes the supportdescribed above.

The method of concentrating and/or purifying aflatoxin in a test sampleaccording to the present invention includes the steps of: preparing asample which has a possibility of containing aflatoxins; applying thesample onto the affinity column described above; and passing an eluentthrough the column to recover an eluate and eluting aflatoxin(s) fromthe antibody or fragment thereof to recover the aflatoxin(s) in theeluate.

The solvent used in the step of applying a sample onto the column may beacetonitrile at a concentration of 1 (v/v) % or more to 20 (v/v) % orless, or methanol at a concentration of 1 (v/v) % or more to 40 (v/v) %or less.

In another aspect of the method of concentrating and/or purifyingaflatoxin according to the present invention, the aflatoxin is at leastone member selected from the group consisting of B1, B2, G1, G2, and M1.

The method of concentrating and/or purifying aflatoxin according to thepresent invention includes the step of allowing a sample containingaflatoxin to be captured by the antibody or fragment thereof which hasbeen carried on the support described above, and then eluting theaflatoxin.

In one aspect of the method of concentrating and/or purifying aflatoxinaccording to the present invention, the aflatoxin is at least one memberselected from the group consisting of B1, B2, G1, G2, and M1.

In the method for concentrating and/or purifying aflatoxin according tothe present invention, the aflatoxin can also be obtained as the totalamount of B1, B2, G1, G2, and M1.

EFFECT OF THE INVENTION

The antibody or fragment thereof of the present invention has an abilityto equally bind to all type of aflatoxins and has organic solventtolerance. In the method of detecting aflatoxins by using this antibodyor fragment thereof, therefore, the total amount or the individualcontents of all aflatoxins even in minute amounts can be accuratelydetected simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an inhibition curve of aflatoxin B2-KLH-immunized mouseantiserum on aflatoxin B2 in indirect competitive ELISA.

FIG. 2 shows inhibition curves of the antibody (AFB2-3-7F3-3) onaflatoxins in direct competitive ELISA.

FIG. 3 shows inhibition curves of the antibody (AFB2-3-7F3-3) onaflatoxin B2 in the presence of methanol in direct competitive ELISA.

FIG. 4 shows inhibition curves of the antibody (AFB2-3-7F3-3) onaflatoxin B2 in the presence of acetonitrile in direct competitiveELISA.

FIG. 5 shows comparison in recovery between the affinity chromatographiccolumn (the present invention) using a gel prepared using the antibody(AFB2-3-7F3-3) and commercially available affinity chromatographiccolumns, where 20% acetonitrile solution containing aflatoxins B1, B2,G1 or G2 was added to each column.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail.

The “aflatoxin” as the subject in this specification refers to at leastone or all of aflatoxins B1, B2, G1, G2, and M1.

Aflatoxin B1 is a compound represented by the following formula:

Aflatoxin B2 is a compound represented by the following formula:

Aflatoxin G1 is a compound represented by the following formula:

Aflatoxin G2 is a compound represented by the following formula:

Aflatoxin M1 is a compound represented by the following formula:

The phases “has an ability to equally bind” and “reacts equally to” meanthat the difference in the reactivity of the antibody or fragmentthereof to B1, B2, G1, G2 or M1 is within ±50% relative to its ability(in terms of 1050 (ng/ml)) to bind to B2.

The term “organic solvent tolerance” used in this specification meansthat in a time point when a test sample is dissolved in an organicsolvent and contacted with an affinity matrix, the antibody or fragmentthereof is not denatured even when allowed to be present in variouskinds of known organic solvents, and particularly means that theantibody or fragment thereof when allowed to be present for at least onehour in 40% methanol or 20% acetonitrile does not change its reactivityto aflatoxins B1, B2, G1, G2, and M1.

When the antibody against aflatoxins is to be produced, aflatoxin B1having a functional group capable of binding to a protein and having thehighest toxicity among the analogs has been generally used as a hapten(immunogen) for immunization. The fact that an antibody showing highreactivity to aflatoxin B1 and having high specificity to aflatoxin B1can be produced by using a derivative of aflatoxin B1 as an immunogen iswell-known.

On the other hand, an antibody showing also high reactivity to otheranalogs, that is, reacting equally to analog aflatoxins B1, B2, G1, G2and M1 (which are at least aflatoxin subjects of measurement) could notbe obtained by the method of using a derivative of aflatoxin B1 as animmunogen, as shown in the Reference Example. This is possibly becausewhen aflatoxin B1 is used as an immunogen, the presence of a double bondbetween C-8 and C-9 in aflatoxin B1, that is, the presence of sp2carbons at positions C-8 and C-9 of the terminal dihydrofuran ring,allows this portion to have a planar structure and simultaneouslyreduces the distance between the carbons at positions 8 and 9, aflatoxinB1. In contrast, aflatoxin B2, having sp3 carbons at positions 8 and 9,has less steric hindrance so that an antibody having a pocket stronglyrecognizing this plane dihydrofuran structure is easily produced, andthis antibody upon binding to aflatoxin B1 enters the deepest part ofthe pocket as the binding site of aflatoxin B1 thereby forming a strongbond through which a strong power to recognize aflatoxin B1 is attained.Aflatoxins B2 and G2 having sp3 carbons at positions C-8 and C-9, on theother hand, are more sterically bulky than aflatoxin B1 because of thepresence of hydrogen atoms protruding vertically at the positions C-8and C-9 from the tetrahydrofuran ring plane, and it is thus supposedthat the antibody cannot deeply enter this pocket, and consequentlyshows a significantly lower power to recognize aflatoxins B2 and G2 thanaflatoxin B1. Also, aflatoxin M1 has a bulky hydroxyl group at position9a and it is thus supposed that an antibody is not able to enter thispocket similar to aflatoxins B2 and G2.

Accordingly, a hapten for producing the antibody meeting the objectivesof the present invention was designed, and for the following reason,aflatoxin B2 was selected as a candidate for the hapten.

It is estimated that since aflatoxin B2 has a single bond between C-8and C-9 and has a more sterically bulky structure around the single bondthan aflatoxin B1, an antibody recognizing aflatoxin B2 can alsorecognize aflatoxin B1. (2) Since aflatoxins G1 and G2 are differentfrom aflatoxins B1 and B2 in their structure in only the vicinity of asite for binding to a carrier protein, it is highly possible that theobtained antibodies do not so much recognize the differencetherebetween. If this hypothesis is correct, it is considered possibleto select, from the obtained antibodies, the antibody reacting equallyto aflatoxins B1, B2, G1, and G2, by using techniques of producingmonoclonal antibodies. (3) Even if aflatoxin B2 is used as an immunogen,the possibility of obtaining those antibodies having high reactivity toaflatoxin M1 is considered lower than to aflatoxins G1 and G2. However,if aflatoxin B2 is used as an immunogen, the possibility of obtainingthose antibodies having a space in which a hydroxyl group of aflatoxinM1 can be accommodated is considered high as compared with aflatoxin B1.Actually, those antibodies obtained with aflatoxin B1 as an immunogen donot always react to aflatoxin M1 but often react to it in a degree aslow as few percent. Accordingly, those antibodies having high reactivityto aflatoxin M1 are considered obtainable with a derivative of aflatoxinB2 as an immunogen rather than with a derivative of aflatoxin B1 as animmunogen.

In view of these working hypotheses, aflatoxin B2 was selected as ahapten in producing those antibodies reacting equally to the respectiveaflatoxin analogs, in order to attempt at producing the antibodieshaving the objective reactivity by techniques of producing monoclonalantibodies.

As the antigen used in the present invention, which is derived fromaflatoxin B2 or derivate thereof, it is possible to conveniently use aconjugate between aflatoxin B2 itself or a compound derived fromaflatoxin B2 by various known methods and a high-molecular weightcompound.

As used herein, the derivative of aflatoxin B2 refers to a compound inwhich a wide variety of known functional groups have been introducedinto aflatoxin B2 to facilitate binding particularly to a high-molecularweight compound. The derivative of aflatoxin B2 in this specificationrefers to a compound having the following skeleton into which afunctional group was introduced into a carbonyl group at position 1 or11 of aflatoxin B2.

A particularly preferable derivative of aflatoxin B2 is a compoundrepresented by the formula (1) and can be obtained by methods including,but not limited to, a method of converting a ketone group at position 1of aflatoxin B2 into a carboxymethoxyimino derivative in accordance witha description in Chu, F. S., Hsia, M-T. S., Sun. P., J. Assoc. Off.Anal. Chem. 60, 791 (1977) .

The compound obtained by the production method as described above can besubjected as necessary to silica gel chromatography andrecrystallization to yield the highly pure objective compound.

The conjugate between the derivative of aflatoxin B2 and ahigh-molecular weight compound may be produced by methods including, butnot limited to, the following method and used as an antigen forimmunization.

Preferable examples of the high-molecular weight compound include suchas keyhole limpet hemocyanin (KLH), ovalbumin (OVA), bovine serumalbumin (BSA), and rabbit serum albumin (RSA).

The binding of the derivative of aflatoxin B2 to the high-molecularweight compound can be achieved by binding a carboxyl group in thederivative to the high-molecular weight compound by methods including,but not limited to, known methods such as an active ester method (A. E.Karu et al.: J. Agric. Food Chem., 42, 301-309 (1994)) and an mixed acidanhydride method (B. F. Erlangeret et al.: J. Biol. Chem., 234,1090-1094 (1954)).

Further, the derivative of aflatoxin B2 to which a labeling substancesuch as an enzyme was bound by the same method as described above can beused in an immunological detection method. The labeling substanceincludes radioisotopes, fluorescence substances and luminescencesubstances. The radioisotopes are not particularly limited, andpreferable examples include such as [125I], [131I], [3H] and [14C]. Theenzymes are not particularly limited and are preferably stable oneshaving large specific activity, and examples include such asβ-galactosidase, β-glucosidase, alkali phosphatase, horseradishperoxidase (referred to hereinafter as “HRP”), and malate dehydrogenase.The fluorescence substances are not particularly limited, and examplesinclude such as fluorescamine and fluorescein isothiocyanate. Theluminescence substances are not particularly limited, and examplesinclude such as luminol, luminol derivatives, luciferin and lucigenin.

In the present invention, the conjugate between a derivative ofaflatoxin B2 and a high-molecular weight compound can be used as anantigen to produce polyclonal or monoclonal antibodies againstaflatoxins.

The polyclonal antibodies can be produced for example by administeringthe conjugate between a derivative of aflatoxin B2 such as the compoundof the formula (1) and a high-molecular weight compound with use of anadministration method such as intraperitoneal injection, intravenousinjection and subcutaneous injection to the site of each animal wherethe antibodies can be produced. In administration, Freund's completeadjuvant or Freund's incomplete adjuvant can also be administered inorder to enhance the ability of the animal to produce antibodies.

The antigen may be administered only once, but is administered usuallyonce every 2 to 6 weeks and about 2 to 10 times in total. The animals inwhich the polyclonal antibodies are produced include such as monkeys,rabbits, dogs, guinea pigs, mice, rats, sheep, goats, and chickens.

The titer of the antibody against the antigen of the present inventionin serum can be measured by a liquid phase method (for example, a methodin which the labeled antigen of the present invention is reacted withantiserum, and then the activity of the label bound to an antibody ismeasured) or a solid phase method (for example, a method in which theantigen of the present invention has been immobilized on the inner wallof each well in a 96-well plate, then a solution having antiserumdiluted appropriately therein is added thereto, an antibody is bound tothe antigen, the solution in each well is washed to remove contaminants,and the amount of the antibody bound to the inner wall of each well ismeasured).

The separation and purification of the polyclonal antibody of thepresent invention are carried out in accordance with a method ofseparating and purifying immunoglobulin. This method includes such assalting-out, alcohol precipitation, isoelectric-point precipitation,electrophoresis, absorption and desorption with ion exchangers (forexample, DEAE), ultracentrifugation, gel filtration, and specificpurification of an antibody by collecting an antibody only with anantigen-bound solid phase or with an active adsorbent such as protein Aor protein G and then dissociating the bonding to obtain the antibody.These techniques may be used alone or in suitable combination thereof.

Production of the monoclonal antibody itself can also be carried outaccording to a usual method (for example, Current Protocol in MolecularBiology, Chapter 11.12-11.13 (2000)). Specifically, a non-human animalsuch as a rabbit, guinea pig, mouse, rat, sheep or goat is immunizedwith the conjugate by a conventional method, then the resulting spleencells are subjected to cell fusion with myeloma cells to preparehybridoma cells which are then screened, and the resulting monoclonalantibody-producing hybridoma can be cultured to give the monoclonalantibody of the present invention (Current Protocols in MolecularBiology edit. Ausubel et al. (1987) Publish. John Wiley and Sons,Section 11.4-11.11).

The monoclonal antibody-producing hybridoma thus obtained is culturedunder predetermined conditions, and while the antibody titer of theproduced antibody is measured, the antibody having the desiredproperties is screened.

The separation and purification of the monoclonal antibody, similar tothe polyclonal antibody, are carried out in accordance with the methodof separating and purifying immunoglobulin. That is, the method includessuch as salting-out, alcohol precipitation, isoelectric-pointprecipitation, electrophoresis, absorption and desorption with ionexchangers (for example, DEAE), ultracentrifugation, gel filtration, andspecific purification of an antibody by collecting an antibody only withan antigen-bound solid phase or with an active adsorbent such as proteinA or protein G and then dissociating the bonding to obtain the antibody.These techniques may be used alone or in suitable combination thereof.

A hybridoma producing the monoclonal antibody described above isprepared by conventional methods including, but not limited to, thefollowing method.

An antigen is mixed with an equal volume of adjuvant and thenadministered intraperitoneally into BALB/c mice. Thereafter, the mouseis subjected periodically to booster immunization. The spleen of a mousehaving an increased antibody titer in serum in collected blood isexcised, and spleen cells were deprived of tissue fragments etc. in aserum-free DMEM medium (Dulbecco's modified Eagle medium), thentransferred to a new medium and suspended completely in the medium. Thecells are washed and mixed with mouse myeloma cells. The cells areprecipitated to remove the supernatant and subjected to cell fusionunder stirring with 50% polyethylene glycol (molecular weight 1500) orthe like. After cell fusion, the cells are suspended in HAT medium orthe like and cultured under suitable conditions. The activity ofantibodies in the culture is examined with ELISA, and cells in a wellwhere the objective antibody has been produced are subjected to limitingdilution, thereby cloning the hybridoma. By cloning, a stable hybridomastrain producing the antibody against aflatoxins is obtained.

The hybridoma of the present invention is cultured in a medium (forexample, DMEM containing 10% fetal bovine serum), and a supernatantobtained by centrifuging the culture can be used as a monoclonalantibody solution. The hybridoma can be injected into the peritonealcavity of the animal from which the hybridoma was derived, to formascites to be used as a monoclonal antibody solution. The antibodysolution can be further purified and concentrated.

In the present invention, the term “fragment” of the antibody refers toan antibody fragment containing an antigen recognition site.

In the present invention, the antibody binding specifically toaflatoxins can be contained to detect aflatoxins easily and usedpreferably as a kit as a detection means and in a method of detectingaflatoxin as described later. Depending on the detection method, the kitalso contains such as a labeled secondary antibody or a labeled haptencompound of aflatoxin, a buffer solution, a detection reagent and/orstandard aflatoxin solutions. A preferable kit can be used in ELISA or adetection method using a gold colloid, and when direct competitive ELISAis used, the kit contains such as an antibody against immobilizedaflatoxin, a support carrying an antibody, and an enzyme-labeled antigenand a detection reagent.

Further, the present invention relates to a method of detectingaflatoxins, which includes using the antibody or detection meansdescribed above. The detection method is not particularly limited aslong as it is a method of utilizing a usual antigen-antibody reaction,and examples of such methods include radioimmunoassay (RIA), enzymeimmunoassay (EIA), a fluorometric or luminometric detection method, anagglutination method, immunoblotting, immunochromatography, and the like(Meth. Enzymol., 92, 147-523 (1983), Antibodies Vol. II IRL Press Oxford(1989)). The labeling means includes such as enzymes, gold colloids,radioisotopes, fluorescence substances and luminescence substances. Theradioisotopes are not particularly limited, and preferable examplesinclude such as [1251], [1311], [3H] and [14C]. The enzymes are notparticularly limited and are preferably stable ones having largespecific activity, and examples include such as β-galactosidase,β-glucosidase, alkali phosphatase, peroxidase, and malate dehydrogenase.The fluorescence substances are not particularly limited, and examplesinclude such as fluorescamine and fluorescein isothiocyanate. Theluminescence substances are not particularly limited, and examplesinclude such as luminol, luminol derivatives, luciferin and lucigenin.Among them, ELISA using β-galactosidase, β-glucosidase, alkaliphosphatase, peroxidase, and malate dehydrogenase, orimmunochromatography using a gold colloid, is particularly preferablefrom the viewpoint of sensitivity and ease.

A method of detection by typical ELISA includes such as indirectcompetitive inhibition ELSA and direct competitive ELISA. For example,aflatoxins can be detected by, for example, direct competitive ELISAusing the polyclonal or monoclonal antibody of the present invention asdescribed below.

(1) The polyclonal or monoclonal antibody of the present invention issolid-phased on a support. The support used is preferably a 96-, 48- or192-well microtiter plate. The antibody may be solid-phased by applying,onto the support, a buffer containing the antibody for immobilizationfollowed by incubation. The concentration of the antibody in the buffersolution is usually from about 0.01 μg/mL to 100 μg/mL. The buffersolution can be a known solution depending on a detection means.

(2) In order to prevent unspecific adsorption of proteins onto thesurface of the solid phase support, the surface of the solid phase onwhich the solid phase antibody was not adsorbed is blocked with aprotein or the like irrelevant to the antibody. As the blocking agent, asolution of BSA or skim milk, commercially available Block Ace(manufactured by Dainippon Sumitomo Pharma Co., Ltd.) or the like can beused. Blocking is carried out by adding the blocking agent to thesupport, incubating it for example at about 4° C. overnight and washingit with a wash fluid. The wash fluid is not particularly limited, andfor example, the same buffer solution as (1) above can be used.

(3) Mixtures in which an enzyme-bound hapten prepared by binding anenzyme to a hapten compound of aflatoxin is added to a sample containingaflatoxin at various concentrations are prepared. Preparation of theEnzyme-Bound Hapten is not Particularly Limited and may be conducted byany method as long as it is a method of binding a hapten compound ofaflatoxin to an enzyme.

(4) The mixture in the step (3) is reacted with the antibody solid phasesupport obtained in the step (2) above. By the competitive inhibitionreaction between aflatoxin and the enzyme-bound hapten, a conjugatebetween aflatoxin or the hapten and the solid phase support is formed.The reaction is carried out for example at about 25° C. for about 1hour. Because aflatoxin is insoluble in water, various organic solventscan be contained in the reaction solution. As the organic solvent, anorganic solvent and its content may be selected in the range such thataflatoxins are dissolved therein and the antigen-antibody reaction isnot inhibited. Specific examples of the solvent include such as methanoland acetonitrile, and a solvent containing acetonitrile at aconcentration of 1 (v/v) % or more to 20 (v/v) % or less, or methanol ata concentration of 1 (v/v) % or more to 40 (v/v) % or less, preferably 1(v/v) % or more to 30 (v/v) % or less, can be used. After the reactionis terminated, the support is washed with a buffer, and the enzyme-boundhapten not bound to the solid phase antibody is removed. By detectingthe amount of the solid phase antibody/enzyme-bound hapten conjugate,the amount of aflatoxins in the sample is determined from a previouslyprepared calibration curve.

(5) A solution of a coloring substrate for reacting with thesupport-bound labeled enzyme is added to detect the absorbance of thereaction solution, whereby the amount of aflatoxins can be calculatedfrom the calibration curve. When a peroxidase is used as the labeledenzyme, a coloring substrate solution containing hydrogen peroxide and3,3′,5,5′-tetramethylbenzidine or o-phenylenediamine for example may beused. Usually, the coloring substrate solution is added to the sample,then the mixture is reacted for about 10 minutes at room temperature,and then sulfuric acid is added to terminate the enzyme reaction. When3,3′,5,5′-tetramethylbenzidine is used, the absorbance of the reactionsolution at 450 nm is measured. When o-phenylenediamine is used, theabsorbance at 492 nm is measured. For correcting the background value,the absorbance at 630 nm is desirably simultaneously measured.

When an alkali phosphatase is used as the labeled enzyme, a method canbe included in which p-nitrophenylphosphoric acid for example is coloredas a substrate, and a solution of NaOH is added to terminate the enzymereaction, and the absorbance of the reaction solution at 415 nm ismeasured.

The reduction rate of the absorbance of the reaction solution in whichaflatoxins were added to and reacted with the antibody, relative to theabsorbance of the reaction solution to which no aflatoxin was added, iscalculated as the inhibition rate. The concentration of aflatoxins inthe sample can be determined by using a calibration curve previouslyprepared from the inhibition rate in the reaction solutions to whichknown concentrations of aflatoxins were added.

In another aspect, detection of aflatoxins can be carried out byindirect competitive ELISA in the following procedures.

The antigen is solid-phased on a support.

The support used is not particularly limited as long as it is a supportused in usual ELISA, and the support is preferably a 96-, 48- or192-well microtiter plate. The antigen may be solid-phased by applying,onto the support, a buffer containing the solid phase antigen followedby incubation. The concentration of the antigen in the buffer solutionis usually from about 0.01 μg/mL to 100 μg/mL. The buffer solution canbe a known solution depending on a detection means.

In order to prevent unspecific adsorption of proteins onto the surfaceof the solid phase support, the surface of the solid phase on which thesolid phase antigen was not adsorbed is blocked with a protein or thelike irrelevant to the antigen. As the blocking agent, a solution of BSAor skim milk, commercially available Block Ace (manufactured byDainippon Sumitomo Pharma Co., Ltd.) or the like can be used. Blockingis carried out by adding the blocking agent to the support, incubatingit for example at about 4° C. overnight and washing it with a washfluid. The wash fluid is not particularly limited, and for example, thesame buffer solution as (1) above can be used.

An aflatoxin-containing sample and a solution of the polyclonal ormonoclonal antibody of the present invention are added to the surface ofthe solid phase treated in (1) and (2) above, and the antibody isreacted competitively with the solid phase antigen and aflatoxin to forma solid phase antigen/antibody conjugate and an antibody conjugateagainst aflatoxin. The reaction can be carried out for example at roomtemperature for about 1 hour. Because aflatoxins are insoluble in water,various organic solvents need to be contained in the reaction solution.As the organic solvent, an organic solvent and its content may beselected in the range such that aflatoxins are dissolved therein and theantigen-antibody reaction is not inhibited. Specific examples of thesolvent include such as methanol and acetonitrile, and a solventcontaining acetonitrile at a concentration of 1 (v/v) % or more to 20(v/v) % or less, or methanol at a concentration of 1 (v/v) % or more to40 (v/v) % or less, preferably 1 (v/v) % or more to 30 (v/v) % or less,can be used.

The amount of the solid phase antigen/antibody conjugate can be detectedby adding an enzyme-labeled secondary antibody (for example, an antibodyrecognizing a mouse antibody). For example, when the mouse monoclonalantibody is used as an antibody against aflatoxin, the antibody againstaflatoxin bound to the support is reacted desirably with an anti-mousegoat antibody labeled with an enzyme (for example, peroxidase oralkaline phosphatase). The reaction may be carried out under the sameconditions as in (3) above. After the reaction, the support is washedwith a buffer solution.

After a solution of a coloring substrate to react with the labelingenzyme of the secondary antibody bound to the support is added, asolution of a coloring substrate to react with the enzyme bound to thesecondary antibody is added similarly to the direct competitive ELISA,and the absorbance of the reaction solution is measured, whereby theamount of aflatoxins therein can be determined from a previous preparedcalibration curve.

In the detection method of the present invention, a sample can bepretreated depending on the detected substance and then subjected todirect competitive ELISA or indirect competitive ELISA. All methods thatcan extract aflatoxins can be used for most of the foods. The solvent inthe extract is changed to methanol or acetonitrile, and then resultingmethanol or acetonitrile solution is diluted with a buffer solution andused as a detection sample. In a simple method, aflatoxins are extractedwith methanol or acetonitrile, then diluted with a buffer solution andused directly as sample.

Further, an affinity column carrying the antibody or fragment thereof ofthe present invention can be used in detection of aflatoxins.

In the present invention, a solid phase adsorbent containing theantibody which binds to all types of aflatoxins and has organic solventtolerance is used thereby enabling detection of the individual amounts,or the total amount, of aflatoxins contained in the test sample as wellas concentration and/or purification of the aflatoxins. Such a method isnot particularly limited, and the following method for example can beused.

The purified antibody or fragment thereof of the present invention canbe used to produce an affinity matrix material (support) by for examplethe following method described by Pharmacia Fine Chemicals. The antibodyused may be a polyclonal antibody, a monoclonal antibody or a fragmentthereof, particularly preferably a monoclonal antibody. In this case, asufficient amount of the monoclonal antibody is dissolved in a bindingbuffer solution, pH 8.3, containing NaHCO3 and NaCl, and the resultingantibody solution is added to Sepharose 4B (Sigma) previously activatedwith cyan bromide by incubation overnight in HCl. After the Sepharose isreacted with the antibody solution, this solid phase adsorbent materialis incubated for a suitable time period in, for example, 1.0Methanolamine, pH 8.5, thereby blocking unbound sites of theantibody-bound gel. An affinity matrix is formed by the monoclonalantibody immobilized on the solid phase adsorbent material and can beused for detection of aflatoxins.

The preferable solid phase adsorbent material includes, but is notlimited to, activated Sepharose 4B gel. Other various materials can beused as a solid phase material and examples thereof include otheragarose gel compositions, dextran, and carbon and silicon granularpreparations including glass plates. Similarly, a method of immobilizingmonoclonal antibodies on each of chemical compositions is also known inthe art and described in various literatures.

The method of detection, isolation, concentration and/or purification ofaflatoxins in a liquid sample in the present invention is exemplified bymethods of, but not limited to, affinity chromatography including thefollowing steps. First, a test sample is contacted with the uniformaffinity matrix having the polyclonal antibody, the monoclonal antibodyor fragment thereof of the present invention immobilized on a solidphase adsorbent material, so that aflatoxins in the test sample arebound to, and supported by, the affinity matrix. The solvent in whichthe test sample was dissolved includes, but is not limited to, such asacetonitrile and methanol. A solvent containing acetonitrile at aconcentration of 1 (v/v) % or more to 20 (v/v) % or less, or methanol ata concentration of 1 (v/v) % or more to 40 (v/v) % or less, preferably 1(v/v) % or more to 30 (v/v) % or less, can be used. Then, an eluent isadded to the affinity matrix in order to release aflatoxins from thepolyclonal antibody, the monoclonal antibody or fragment thereof of thepresent invention. This eluent includes, but is not limited to, such asacetonitrile and methanol. Then, whether aflatoxins are present or notin the eluate recovered from the affinity matrix, and which types ofaflatoxins are contained in what amount, can be determined for exampleby HPLC, etc.

The method of concentration and purification of aflatoxins in a liquidsample according to the present invention is exemplified by methodsincluding, but not limited to, the following method using the polyclonalantibody, the monoclonal antibody or fragment thereof of the presentinvention. First, a test sample is contacted with the uniform affinitymatrix having the polyclonal antibody, the monoclonal antibody orfragment thereof of the present invention immobilized on a solid phaseadsorbent material, so that aflatoxins in the test sample are bound to,and supported by, the affinity matrix. Then, contaminants are removed bywashing. Then, an eluent is added to the affinity matrix in order torelease aflatoxins from the polyclonal antibody, the monoclonal antibodyor fragment thereof of the present invention. In this manner, aflatoxinsin the liquid sample can be concentrated in an immunologically lesscontaminated state several thousand times to several ten thousand timeshigher than the concentration in the original sample. Accordingly, evenaflatoxins present in very small amounts in a sample can be concentratedto a much higher degree than in other concentration methods using noneof the antibody. In the concentrate so obtained, the content ofcontaminants or the like interfering with quantification is low. Thesolvent used in dissolving the test sample to load it onto the affinitymatrix includes, but is not limited to, such as acetonitrile andmethanol. A solvent containing acetonitrile at a concentration of 1(v/v) % or more to 20 (v/v) % or less, or methanol at a concentration of1 (v/v) % or more to 40 (v/v) % or less, preferably 1 (v/v) % or more to30 (v/v) % or less, can be used.

An important feature of the polyclonal and monoclonal antibody againstaflatoxin according to the present invention is that the antibody has ahigh ability (affinity) to bind not only to aflatoxin B2 antigen butalso to B1, G1, G2 and M1 equally. The antibody of the present inventionhas organic solvent tolerance superior to that of usual existinganti-aflatoxin B1 monoclonal antibodies. Accordingly, the solvent usedin dissolving a test sample to contact it with the affinity matrix canbe an organic solvent having a relatively high concentration.

Hereinafter, the present invention is described in more detail withreference to the Examples, but these examples are not intended to limitthe technical scope of the present invention. Those skilled in the artcan easily modify and alter the present invention based on thedescription of this specification, and such modifications andalterations are contained in the technical scope of the presentinvention.

Example 1 Synthesis of the Compound (Aflatoxin Hapten) of Formula (1)

8.2 mg (26.1 μmol) of aflatoxin B2 (Wako Pure Chemical Industries, Ltd.)was dissolved in 6.3 ml solution of pyridine:methanol:water (1:4:1), and10.3 mg (93.9 μmol) of aminooxyacetic acid hemihydrochloride was addedthereto and heated under reflux for 2 hours. This reaction mixture wasconcentrated, and the residues were purified through a silica gel column(chloroform:methanol=9:1) to give aflatoxin B2 hapten, 9.1 mg (yield90%), in the form of white powder.

The Rf value of the resulting B2 hapten on TLC (silica gel 70F254 platemanufactured by Wako Pure Chemical Industries, Ltd.; developing solvent,chloroform:methanol=9:1) was 0.70 for aflatoxin B2 and 0.54 for thehapten.

Example 2 Preparation of an Immunogen

A conjugate between keyhole limpet hemocyanin (KLH) and the hapten offormula (1) obtained in Example 1 was prepared as an immunogen by theactive ester method.

3.3 mg of the aflatoxin B2 hapten produced in Example 1, 2.2 mg ofN-hydroxysuccinimide, and 3.7 mg of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride weredissolved in 0.5 ml of dimethylsulfoxide, and the resulting solution wasstirred for 1.5 hours at room temperature in a dark place to prepare anaflatoxin B2 hapten solution. Separately, 10 mg of KLH was added to 1 mlof 0.1 M borate buffer solution, pH 8.0, and 248 μl of the activatedaflatoxin B2 hapten solution was added dropwise thereto and stirred for1.5 hours at room temperature in a dark place. After the reaction wasterminated, the reaction mixture was dialyzed against a physiologicalphosphate buffer solution (10 mM phosphate buffer solution containing150 mM NaCl and being adjusted to pH 7.0) at 4° C. for 2 days and thenstored at −40° C. Similarly, a conjugate between the aflatoxin haptenand BSA was prepared. The conjugate between the aflatoxin hapten and KLHobtained in this manner was used as an immunogen.

Example 3 Preparation of a Monoclonal Antibody-Producing Hybridoma

The immunogen prepared in Example 2 was dissolved at 2 mg/mL in PBS,mixed with an equal volume of complete adjuvant (trade name: Freund'scomplete adjuvant; FCA) and then emulsified, and 100 μL of the resultingemulsion was administered intraperitoneally to a 6- to 7-week-old femaleBALB/c mouse. In a similar manner, 100 μL of 0.5 mg/mL immunogen mixedwith an equal volume of incomplete adjuvant (trade name: Freund'sincomplete adjuvant: FICA) was administered as booster at 2-weekintervals. After immunization was conducted 4 times, blood was collectedfrom eye ground, and it was confirmed by the indirect competitive ELISAthat the antibody titer in serum had been sufficiently increased.

Detection of Aflatoxins by the Indirect Competitive ELISA Method

The conjugate between the aflatoxin hapten and BSA obtained in Example 2was diluted to 1 μg/mL with 10 mM sodium phosphate buffer containing 150mM NaCl, dispensed in a volume of 100 μL/well into a 96-well microplateand then solid-phased by leaving it overnight at 4° C. Then, the fluidin each well was removed by suction, and then 10 mM sodium phosphatebuffer containing 0.4% BSA and 150 mM NaCl and being adjusted to pH 7.0was dispensed in a volume of 300 μL/well, and each well was blocked byleaving it overnight at 4° C., followed by removing the blockingsolution by suction.

(2) 0, 100, 250, 500, 1000, 2500, 5000, and 10000 ng/ml standardsolutions of aflatoxins B1, B2, G1, G2 and M1 dissolved in methanol wereadded at 1% to 10 mM sodium phosphate buffers containing 0.2% BSA and150 mM NaCl and being adjusted to pH 7.0, to prepare solutionscontaining each aflatoxin at 0.0, 1.0, 2.5, 5.0, 10, 25, 50 and 100ng/mL, respectively. Dilution solutions of each aflatoxin were dispensedin a volume of 50 μL/well into the plate on which the conjugate betweenthe aflatoxin hapten and BSA prepared in (1) had been solid-phased. Adilution obtained by diluting the previously obtained antiserum20,000-fold with 10 mM sodium phosphate buffer containing 0.2% BSA and150 mM NaCl and being adjusted to pH 7.0 was further added in a volumeof 50 μL/well to the plate which was then left at 25° C. for 1 hour andsubjected to aflatoxin competitive inhibition reaction.

(3) The conjugate between HRP (horseradish peroxidase) and an anti-mouseIgG sheep antibody (ICN Inc.) was diluted 4000-fold with 10 mM sodiumphosphate buffer containing 0.2% BSA and 150 mM NaCl and being adjustedto pH 7.0 to prepare a secondary antibody dilution.

(4) The well reacted in (2) was washed 3 times with 10 mM sodiumphosphate buffer containing 150 mMNaCl, and then the secondary antibodydilution was dispensed in a volume of 100 μL/well to the plate andreacted by leaving it at 25° C. for 1 hour.

Each well after being reacted in (4) above was washed 3 times with 10 mMsodium phosphate buffer containing 150 mM NaCl, and 100 μl of a TMBsubstrate solution (0.1 N sodium acetate solution (pH 5.5) to which 100μg/mL of 3,3′,5,5′-tetramethylbenzidine and 0.006% of hydrogen peroxidehad been added) was added to each well and incubated at 25° C. for 10minutes, and then 100 μL of 1 N sulfuric acid was added to each well toterminate the coloring reaction, and the absorbance at 450 nm wasmeasured with a microplate reader. The results of aflatoxin B2 in thisexample are shown in FIG. 1.

Confirmation of a Monoclonal Antibody

A mouse having antibody titer increased sufficiently in blood wasfinally immunized (10 μg/mouse). Three days thereafter, the spleen wasexcised from the mouse and subjected to cell fusion.

After a superfluous tissue section was removed from the excised spleenin a serum-free DMEM medium (Dulbecco's modified Eagle medium), cellswere removed completely from the spleen and suspended in the medium. Inorder to precipitate suspended large tissue sections, the sample wasleft for 5 minutes, and the cell suspension was collected in acentrifuge tube and centrifuged at 1500 rpm, and the supernatant wasremoved by suction, and a fresh serum-free DMEM was added thereto tosuspend the cells. This operation was carried out twice.

Previously cultured myeloma cells (P3X63Ag8.653) were recovered,centrifuged to remove the supernatant, and resuspended repeatedly twicein a serum-free DMEM medium.

The respective cells were counted and mixed such that the ratio of thespleen cells to the myeloma cells became 10:1 to 7.5:1, and the mixedcells were centrifuged at 1500 rpm for 5 minutes and the supernatant wasremoved by suction.

While the centrifuge tube was stirred vigorously, 2 mL of 50%polyethylene glycol (molecular weight 1500) was added over about 60seconds. Then, about 10 mL of serum-free DMEM was added over 3 to 4minutes while stirring.

The centrifuge tube was centrifuged at 1000 rpm for 5 minutes to removethe supernatant completely, and the spleen cells were suspended at2.5×106 cells/mL in HT medium (DMEM medium supplemented withhypoxanthine, thymidine and 10% fetal bovine serum) and dispensed in avolume of 100 μL/well into a 96-well culture plate, and culture wasinitiated at 37° C. in 8% carbon dioxide gas under humidifiedconditions.

On the next day, HAT medium (DMEM medium supplemented with hypoxanthine,thymidine, aminopterin, 10% fetal bovine serum) was added in a volume ofabout 40 μL/well, and the cells were observed until the myeloma cellsperished and a colony of the hybridoma cells was formed, and thereafter,HT medium was added while the cells were continuously observed.

Ten days after culture was initiated, the culture was collected, and awell in which the antibody against aflatoxin had been produced wasselected by the indirect competitive ELISA method and cultured in anincreasing scale in 96 wells, 48 wells and 24 wells sequentially.

Cloning was carried out by limiting dilution in the 24-well stage, and ahybridoma strain producing an AFB2-3-7F3-3 monoclonal antibody againstaflatoxin was obtained. The resulting hybridoma strain has beendomestically deposited under Accession No. FERM P-21126 withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, Japan. This hybridoma strain has alsobeen internationally deposited under Accession No. FERM ABP-10931 sinceNov. 5, 2007, with International Patent OrganismDepositary, NationalInstitute of Advanced Industrial Science and Technology.

Hybridoma screening was carried out by using, as an indicator, areactivity being less than 1 ppb and being exhibited almost equallytoward aflatoxins B1, B2, G1 and G2. By further screening hybridomas byusing, as an indicator, a reactivity being as high as possible toaflatoxin B1 in the presence of varying concentrations of acetonitrileand methanol, a plurality of antibody-producing cells showing similarreaction characteristics could also be selected besides the AFB2-3-7F3-3antibody-producing cells.

Example 4 Preparation of a Monoclonal Antibody

The AFB2-3-7F3-3 antibody-producing hybridoma strain obtained in Example3 was cultured in DMEM containing 10% fetal bovine serum, and about2×106 cells were injected intraperitoneally to Balb/c female Retiremouse, and then the ascites fluid was collected. The resulting ascitesfluid was applied onto a protein G column to purify IgG. The subclass ofthe obtained monoclonal antibody was IgG1 with λ, chain as light chain.

On the other hand, the AFB2-3-7F3-3 antibody-producing cells werecultured at 37° C. in the presence of 5% CO2 in Dulbecco's MEM mediumsupplemented with 10% fetal bovine serum, and were confluentlyproliferated. The cells were centrifuged at 1500 rpm to give the culturesupernatant. The culture supernatant was diluted 50-fold with 10 mMsodium phosphate buffer containing 0.2% BSA and 150 mM NaCl and beingadjusted to pH 7.0, and then subjected to an indirect competitive ELISAmethod.

Example 5 Detection of Aflatoxins by an Indirect Competitive ELISAMethod

The conjugate between the aflatoxin hapten and BSA obtained in Example 2was diluted to 1 μg/mL with 10 mM sodium phosphate buffer containing 150mM NaCl and dispensed in a volume of 100 μL/well into a 96-wellmicroplate and then solid-phased by leaving it overnight at 4° C. Then,the fluid in each well was removed by suction, and then 10 mM sodiumphosphate buffer containing 0.4% BSA and 150 mM NaCl and being adjustedto pH 7.0 was dispensed in a volume of 300 μL/well, and each well wasblocked by leaving it overnight at 4° C., followed by removing theblocking solution by suction.

0, 10, 25, 50, 100, 250, 500, and 1000 ng/ml standard solutions ofaflatoxins B1, B2, G1, G2 and M1 dissolved in methanol were added at 1%to 10 mM sodium phosphate buffers containing 0.2% BSA and 150 mM NaCland being adjusted to pH 7.0, to prepare solutions containing eachaflatoxin at 0.0, 0.1, 0.25, 0.50, 1.0, 2.5, 5.0, and 10.0 ng/ml,respectively. Dilution solutions of each aflatoxin were dispensed in avolume of 50 μL/well into the plate on which the conjugate between theaflatoxin hapten and BSA prepared in (1) had been solid-phased. Adilution obtained by diluting a culture of the monoclonal antibodyAFB2-3-7F3-3-producing cells obtained in Example 4, 50-fold with 10 mMsodium phosphate buffer containing 0.2% BSA and 150 mM NaCl and beingadjusted to pH 7.0 was further added in a volume of 50 μL/well to theplate which was then left at 25° C. for 1 hour and subjected toaflatoxin competitive inhibition reaction.

Hereinafter, the same procedures as in Example 3 were conducted.

Standard inhibition curves of the monoclonal antibody AFB2-3-7F3-3solution on aflatoxins B1, B2, G1, G2 and M1 by indirect competitiveELISA are shown in FIG. 2. The IC50 values for all types of aflatoxinswere 0.26 for B1, 0.34 for B2, 0.34 for G1, 0.34 for G2, and 0.4 for M1by indirect competitive ELISA using the monoclonal antibody AFB2-3-7F3-3solution, and therefore, the reactivity of the antibody to B1, B2, G1,G2 and M1 was within ±50% relative to B2 in term of IC50 values, thusshowing equal detection sensitivity among the aflatoxins.

The monoclonal antibodies obtained in other hybridomas obtained inExample 3 were also examined in the same experiment. As a result, themonoclonal antibody AFB2-2-5E10-1 solution for example showed IC50values for all types of aflatoxins as follows: 0.33 for B1, 0.44 for B2,0.43 for G1, 0.57 for G2, and 0.55 for M1, and the AFB2-3-4E4-1 solutionshowed the following IC50 values: 0.3 for B1, 0.44 for B2, 0.43 for G1,0.53 for G2, and 0.58 for M1.

These values indicate that the antibodies of the present invention havean excellent ability to bind to all of aflatoxins B1, B2, G1, G2 and M1.There are those previously existing monoclonal antibodies againstaflatoxins which are induced against B1 as a hapten, but any of suchmonoclonal antibodies are not those which in comparison with theirability to bind to other types of aflatoxins, “have an ability toequally bind” referred to in this specification. In JP-A 63-219394, forexample, it is attempted to obtain antibodies raised against B1 ashapten and specific to B1, and therefore, their ability to bind to G1 isreduced significantly in comparison with their ability to bind to B1 orB2. For example, an antibody referred to in Table 1 as AF-1 has theminimum difference in its binding ability, but nevertheless, its abilityto bind to G1 is about 1/10 or less relative to its ability to bind toB1 or B2. On the other hand, there is an example of a producedmonoclonal antibody useful for detecting all aflatoxins, but in theExamples in JP-A 4-360695, such a monoclonal antibody showedcrossreactivities of 0.44 ng/ml, 2.1 ng/ml, 2.4 ng/ml and 5.2 ng/ml interms of IC50 for aflatoxins B1, B2, G1 and G2, respectively.

From the foregoing results, it can be seen that analysis of the totalamount of all types of aflatoxins and analysis among the aflatoxinanalogs become possible with high accuracy by indirect competitive ELISAmethod using the monoclonal antibody obtained with use of the aflatoxinhapten compound of the present invention.

Example 6 Methanol Tolerance of the Antibody

The methanol tolerance of the monoclonal antibody AFB2-3-7F3-3 wasexamined. 0%, 20%, 40%, 60%, 80% and 100% aqueous methanol solutionswere prepared, and each aqueous methanol solution was mixed at a ratioof 19/1 with 10 mM sodium phosphate buffer containing 0.2% BSA and 150mM NaCl and being adjusted to pH 7.0, and further 0, 10, 25, 50, 100,250, 500 and 1000 ng/ml standard solutions of aflatoxin B2 were added at1% to the aqueous methanol solutions each having the predeterminedconcentration, to prepare solutions containing aflatoxin B2 at aconcentration of 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 ng/mL,respectively. The solution containing methanol at each concentration andaflatoxin B2 at each concentration was added in a volume of 50 μL/wellto the plate on which the conjugate between the aflatoxin hapten and BSAprepared in (1) in Example 3 had been solid-phased.

On the other hand, a culture of the monoclonal antibodyAFB2-3-7F3-3-producing cells was diluted 2.5-fold with 10 mM sodiumphosphate buffer solution containing 0.2% BSA and 150 mM NaCl and beingadjusted to pH 7.0, and added in a ratio of 19:1 to 0%, 20%, 40%, 60%,80% and 100% methanol solutions respectively to prepare antibodydilutions.

The antibody dilution was added in a volume of 50 μL/well to the wellhaving the same methanol concentration on the plate into which theaflatoxin B2 solutions having the predetermined concentrations had beendispensed, and the antibody was reacted by leaving it at 25° C. for 1hour. Thereafter, the reaction was carried out as shown in (3), (4) and(5) in Example 3 to determine inhibition curves in the methanolconcentrations in the reaction solutions.

The results are shown in FIG. 3. The aflatoxin antibody AFB2-3-7F3-3showed an inhibition curve which is not disturbed even in 38% methanol,and had high tolerance to methanol. When aflatoxins present in foods areextracted, methanol is a very excellent solvent to be generally used,and it is shown that AFB2-3-7F3-3 having high tolerance to methanol isextremely useful as an antibody for detection of aflatoxins.

Example 7 Acetonitrile Tolerance of the Antibody

The acetonitrile tolerance of the monoclonal antibody AFB2-3-7F3-3 wasexamined. 0%, 20%, 40%, 60%, 80% and 100% aqueous acetonitrile solutionswere prepared, and 0, 10, 25, 50, 100, 250, 500 and 1000 ng/ml standardsolutions of aflatoxin B2 were added at 1% to the aqueous acetonitrilesolutions each having the predetermined concentration, to preparesolutions containing aflatoxin B2 at a concentration of 0, 0.1, 0.25,0.5, 1.0, 2.5, 5.0 and 10.0 ng/ml, respectively. The solution containingacetonitrile at each concentration and aflatoxin B2 at eachconcentration was added in a volume of 50 μL/well to the plate on whichthe conjugate between the aflatoxin hapten and BSA prepared in (1) inExample 3 had been solid-phased.

On the other hand, a culture of the monoclonal antibodyAFB2-3-7F3-3-producing cells was diluted 50-fold with 10 mM sodiumphosphate buffer solution containing 0.2% BSA and 150 mM NaCl and beingadjusted to pH 7.0, and dispensed in a volume of 50 μl/well to each wellof the previous plate. The antibody was reacted by leaving it at 25° C.for 1 hour, and thereafter, the reaction was carried out as shown in(3), (4) and (5) in Example 3 to determine inhibition curves in 0%, 10%,20%, 30%, 40% and 50% acetonitrile concentrations in the reactionsolutions.

The results are shown in FIG. 4. The aflatoxin antibody AFB2-3-7F3-3showed an inhibition curve which is not disturbed even in 20%acetonitrile, and had high tolerance to acetonitrile. When aflatoxinspresent in foods are extracted, acetonitrile is a very excellent solventto be generally used, and it is shown that AFB2-3-7F3-3 having hightolerance to acetonitrile is extremely useful as an antibody fordetection of aflatoxins.

Example 8 Preparation of Gel

1 mL of NHS-activated Sepharose 4FF (GE Healthcare Bio Science) waswashed 3 times with 10 ml of 1 mM cold hydrochloric acid. 10 ml of theantibody obtained in Example 4 (antibody solution diluted with 10 mMsodium phosphate buffer solution containing 0.5 mg/ml antibody, 0.2% BSAand 150 mM NaCl and being adjusted to pH 7.0) was added thereto, and thegel was stirred at room temperature for 4 hours and then washed 3 timeswith 10 ml each of a monoethanolamine buffer solution, a sodium acetatebuffer solution and a monoethanolamine buffer solution in this order. 10ml of a monoethanolamine buffer solution was added thereto, and the gelwas stirred at room temperature for 3 hours and washed 3 times with 10ml each of a sodium acetate buffer solution, a monoethanolamine buffersolution, a sodium acetate buffer solution and a phosphate buffersolution in this order.

Example 9 Preparation of an Affinity Column for Aflatoxins

An empty column was packed with 0.2 mL of the gel prepared in Example 8and then washed twice with 3 ml of 10 mM sodium phosphate buffercontaining 150 mM NaCl.

Example 10 Experiment of Addition onto and Recovery with the AffinityColumn for Aflatoxins

Onto this column was added 10 ml of each of 2% acetonitrile solutionseach containing aflatoxins B1, B2, G1 and G2 at 6 concentrations, thatis, 5 ng, 10 ng, 25 ng, 50 ng, 100 ng and 200 ng. Then, this column waswashed twice with 3 ml of 10 mM sodium phosphate buffer solutioncontaining 150 mM NaCl and twice with 3 ml water and eluted 3 times with1 ml of 100% acetonitrile.

Then, the resulting eluate was analyzed with HPLC, and each type ofaflatoxins B1, B2, G1 and G2 in the eluate was measured. The HPLCconditions are as follows: that is, column: ODS column (4.6×150 mm, 5μm), mobile phase: acetonitrile-methanol-0.2 M ammonium acetate buffer(pH 5.0)-water (1:3:0.5:5.5), flow rate 1.0 ml/min, column temperature40° C., detector: fluorescence detector (excitation wavelength 360 nm,fluorescence wavelength 450 nm), injection volume: 100 μl, and aphotochemical reactor (Aura Industries Inc.) was arranged between thecolumn and the detector.

The recovery (%) is as shown in Table 1.

TABLE 1 Aflatoxins 5 ng 10 ng 25 ng 50 ng 100 ng 200 ng G2 107 100 95 9489 46 G1 95 95 94 92 89 54 B2 108 98 91 88 88 61 B1 99 94 89 87 88 72(The unit of numerical values in the table is %)

From Table 1, it was found that all types of aflatoxins B1, B2, G1 andG2 was able to be almost accurately recovered in the range of from 5 ngto 100 ng.

Example 11

The affinity column using the monoclonal antibody of the presentinvention, prepared in Example 10, and commercially available affinitycolumns for aflatoxins, were used in a comparative experiment. 10 mleach of aflatoxin standard preparations (that is, 20% acetonitrilesolutions each containing 50 ng each of aflatoxins B1, B2, G1 and G2)was added to the column prepared in Example 10 and commercial columns A,B and C, respectively. Each of these columns was washed twice with 3 mlof 10 mM sodium phosphate buffer containing 150 mM NaCl and twice with 3ml water and eluted 3 times with 1 ml of 100% acetonitrile. FIG. 5 showsthe recoveries of aflatoxin types B1, B2, G1 and G2 from each column.

From FIG. 5, it can be seen that the recovery from the commercialcolumns varies among the aflatoxin analogs, while the recovery from theaffinity column having the monoclonal antibody of the present inventionimmobilized thereon hardly varies among the analogs, and thus has idealproperties of concentrating and purifying all aflatoxin analogs.Moreover, a solvent containing an organic solvent at high content, suchas 20% acetonitrile, can also be used in the affinity column having themonoclonal antibody of the invention immobilized thereon.

From the foregoing, the monoclonal antibody obtained in the presentinvention, and an affinity column capable of concentrating and purifyingaflatoxins by using the antibody, can be used not only in highlyaccurate quantification of aflatoxins but also in quantification of theindividual contents of aflatoxin analogs and the total amount ofaflatoxin analogs.

Reference Example

For aflatoxin B1, an aflatoxin B1 hapten was obtained by the method foraflatoxin B2 shown in Example 1. This hapten compound was used toprepare an immunogen by the method shown in Example 2 and this immunogenwas used to immunize a mouse by the method shown in Example 3.Similarly, a rabbit was also immunized. The immunogen prepared inExample 2 was dissolved at 0.4 mg/mL in PBS, mixed with an equal volumeof complete adjuvant (trade name: Freund's complete adjuvant; FCA) andthen emulsified, and 2 mL of the resulting emulsion was administeredintracutaneously to a 2.5 to 3.0 kg female rabbit (Japanese whitespecies). In a similar manner, the immunogen mixed with an equal volumeof incomplete adjuvant (trade name: Freund's incomplete adjuvant: FICA)was administered as booster 4 times at 2-week intervals. One week afterfinal immunization was conducted, whole blood was collected, and theantibody titer in serum was confirmed by indirect competitive ELISA tobe sufficiently increased. The antibody titer in serum was confirmed byindirect competitive ELISA. The results are shown in Table 2.

TABLE 2 Ability of antiserum to bind to aflatoxin analogs Immunizedanimal IC50 ng/ml (%) Mouse Rabbit Aflatoxin B1 0.07 (2163) 0.07 (2429)Aflatoxin B2 1.5 (100) 1.7 (100) Aflatoxin G1 0.6 (250) 0.63 (270) Aflatoxin G2 16 (9)   18 (9)   Aflatoxin M1 11 (14)  14 (12) 

From the results in Table 2, it can be seen that as a result ofimmunization of the animal with the immunogen obtained using thederivative of aflatoxin B1 as a hapten, the resulting antiserum, whetherin the mouse or the rabbit, gives a particularly high activity to bindto aflatoxin B1, gives a lower activity to bind to aflatoxin G1 than toB1, and shows an only lower activity to bind to aflatoxins G2 and M1.

Sheet copy (Attention: Original is an electronic data)(This sheet does not construct a part of International application andnot includes number of sheets of International application)

0-1 Form PCT/RO/134 (SAFE) INDICATIONS RELATING TO DEPOSITED 0-1-1MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCTRule13-2) JPO-PAS weremade by right 342 0-2 International Application Number 0-3 Applicant'sor agent's PCT07050HR file reference 1 The indications made below relateto the deposited microorganism or other biological material referred toin the description 1-1 Paragraph number 0017 1-3 Identification ofDeposit 1-3-1 Name of depositary IPOD International Patent institutionOrganism Depositary National Institute of Advanced Industrial Scienceand Technology (IPOD) 1-3-2 Address of depositary AIST Tsukuba Central6, 1-1, institution Higashi 1-chome Tsukuba-shi, Ibaraki-ken 305-8566Japan 1-3-3 Date of deposit Nov. 5, 2007(05.11.2007) 1-3-4 AccessionNumber IPOD FERM ABP-10931 1-5 Designated States for All DesignatedStates which indications are made

For Receiving Office use only 0-4 This sheet was received with theinternational application (Yes/No) 0-4-1 Authorized officer ForInternational Bureau use only 0-5 The date when This sheet was receivedby the International Bureau 0-5-1 Authorized officer

1. An antibody or fragment thereof, which is obtained by using anantigen derived from aflatoxin B2 or derivative thereof, has an abilityto equally bind to all aflatoxins B1, B2, G1, G2, and M1, and hasorganic solvent tolerance.
 2. The antibody or fragment thereof accordingto claim 1, wherein the derivative of aflatoxin B2 is a compound havinga structure represented by the following formula (1):


3. The antibody or fragment thereof according to claim 1, wherein theantigen derived from the derivative of aflatoxin B2 is a conjugatebetween the compound of the formula (1) and a high-molecular compound.4. The antibody or fragment thereof according to claim 1, wherein theantibody is a polyclonal antibody.
 5. The antibody or fragment thereofaccording to claim 1, wherein the antibody is a monoclonal antibody. 6.The antibody or fragment thereof according to claim 5, wherein themonoclonal antibody is AFB2-3-7F3-3.
 7. A hybridoma producing theantibody or fragment thereof according to claim
 5. 8. The hybridomaaccording to claim 7, which has been internationally deposited underAccession No. FERM ABP-10931.
 9. A method of immunologically detectingaflatoxins, which comprises using the antibody or fragment thereofaccording to claim
 1. 10. The method of immunologically detectingaflatoxins according to claim 9, which can detect the amount of one ormore members selected from the group consisting of aflatoxins B1, B2,G1, G2, and M1 or the total amount of all the members.
 11. The method ofimmunologically detecting aflatoxins according to claim 9, which furthercomprises using a solid phase adsorbent.
 12. A support comprising asolid phase adsorbent and the antibody or fragment thereof of claim 1immobilized on the solid phase adsorbent.
 13. The support according toclaim 12, wherein the solid phase adsorbent is Sepharose.
 14. Anaffinity column comprising the support according to claim
 12. 15. Amethod of concentrating and/or purifying aflatoxin in a test sample,comprising the steps of: preparing a sample which may containaflatoxins; applying the sample onto the affinity column of claim 14;and passing an eluent through the column to recover an eluate andeluting the aflatoxin(s) from the antibody or fragment thereof torecover the aflatoxin(s) in the eluate.
 16. The method of concentratingand/or purifying aflatoxin according to claim 15, wherein the solventused in the step of applying a sample onto the column is acetonitrile ata concentration of 1 (v/v) % or more to 20 (v/v) % or less, or methanolat a concentration of 1 (v/v) % or more to 40 (v/v) % or less.
 17. Themethod of concentrating and/or purifying aflatoxin according to claim15, wherein the aflatoxin is at least one member selected from the groupconsisting of B1, B2, G1, G2, and M1.
 18. A method of concentratingand/or purifying aflatoxin, which comprises allowing anaflatoxin-containing sample to be captured by the antibody or fragmentthereof which has been carried on the support of claim 12, and theneluting the aflatoxin.
 19. The method of concentrating and/or purifyingaflatoxin according to claim 18, wherein the aflatoxin is at least onemember selected from the group consisting of B1, B2, G1, G2, and M1. 20.The method for concentrating and/or purifying aflatoxin according toclaim 15, wherein the aflatoxin is the total amount of B1, B2, G1, G2,and M1.